1. Field of the Invention
The present invention concerns a method to inactivate viruses in biological compositions, for example, in whole blood or red blood cell concentrates, without incurring substantial disruption or inactivation of cells, for example, without adversely affecting red blood cell structure or function, by using a photoactive compound, for example, a phthalocyanine, together with light exposure and variations thereon.
2. Description of Related Art
Nature of the Concept
Substantial progress has been made in reducing the viral infectivity of whole blood and its components through improved donor selection and donor blood screening procedures. Despite this progress, there is a continued risk of transmission of viruses including hepatitis viruses and human immunodeficiency viruses (HIV) by whole blood and blood products.
The risk of transmission of certain viruses (e.g., hapatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV)) has been considerably reduced and possibly eliminated in coagulation factor concentrates through the application of virucidal procedures during the course of manufacture (Prince, A. M., Horowitz, B., Horowitz, M. S., Zang, E., "The Development of Virus-Free Labile Blood Derivatives--A Review", Eur. J. Epidemiol., 1987; 3:103-118 and Mannucci, P. M., Colombo, M., "Virucidal Treatment of Clotting Factor Concentrates", The Lancet, 1988;782-785). However, when treating coagulation factor concentrates, some viruses (e.g., parvovirus) may remain infectious. In addition, the development of virucidal processes applicable to cell components, i.e., blood cell fractions such as red blood cells or platelets, has been slow, both because cells are more fragile than proteins, and cells serve to harbor and protect virus against inactivation. Nonetheless, if virus transmission by whole blood or blood components is to be eliminated, effective virus removal or potent virucidal methods applicable to blood cells will be required. Since both red blood cells or platelets are non-replicating, approaches directed toward nucleic acid modification might offer the required specificity.
It is important to recognize in assessing virucidal procedures for cell-containing solutions that virus will be present in multiple forms: virus free of the cell; formed virus associated with the cell; functional, but unpackaged viral nucleic acid within the cell; and viral nucleic acid integrated into the cell genome. Each form should be considered infectious and capable of causing viral disease in vivo. Virucidal methods which inactivate virus in one form, e.g., cell-free virus, may not inactivate virus in other forms, e.g., cell-associated forms. Additionally, the presence of cells is known to inhibit the action of both physical and chemical approaches to virus inactivation. Cells compete for added virucidal reagents and absorb radiation which otherwise would be virucidal. Thus, for example, while ultraviolet irradiation is highly virucidal in salt solutions or in dilute protein solution, the degree of virucidal activity is incomplete when treating cell-containing solutions. Furthermore, in this context, it is not sufficient to inactivate virus alone; rather, it is necessary to do so with sufficient vigor that viral infectivity is eliminated without deleterious effects to the valuable cell components, e.g., red blood cells.
Most virucidal procedures which have been developed, e.g., pasteurization or solvent/detergent treatment, cannot be applied to blood cell preparations without damaging the cells and rendering them unfit for transfusion.
Heretofore, it has not been possible to prepare virus sterilized forms of whole blood or red cell concentrates or platelet concentrates where at least 10.sup.4 infectious units (ID.sub.50) and preferably .gtoreq.10.sup.6 ID.sub.50 of both both intracellular and extracellular virus were inactivated without adversely affecting the cells and/or without using highly toxic agents.
Phthalocyanines
While there has been growing interest in the use of phthalocyanines for the treatment of cancerous cells (Rosenthal, I. and Ben Hur, E., "Phthalocyanines in Photobiology", Lezhoff C. C. and Lever A. B. P. eds., Phthalocyanines, VCH Publishers, Inc., New York, New York, 1989, 393-425), phthalocyanines are generally thought of as being hemolytic, making applicants' results herein all the more surprising. For example, Ben-Hur and Rosenthal ("Photohemolysis of Human Erthrocytes Induced by Aluminum Phthalocyanine Tetrasulfonate", Cancer Lett., 30: 321-327, 1986) studied the photohemolysis of human red blood cells induced by aluminum phthalocyanine tetrasulfonate. Substantial (20-100%) hemolysis was induced by treatment with 2.5 to 10 .mu.M AlPcS.sub.4 at all light fluences .gtoreq.40 KJ/m.sup.2 (.gtoreq.4 J/cm.sup.2). Ben-Hur and Rosenthal did not address the problem of virus kill. Similarly, Sonoda, Krishna and Riesz ("The Role of Singlet Oxygen in the Photohemolysis of Red Blood Cells Sensitized by Phthalocyanine", Photochem. Photobiol., 46: 625-631, 1987) studied the photohemolysis induced by each of several phthalocyanine derivatives. Aluminum and zinc phthalocyanines were each hemolytic, while free (no metal) phthalocyanine or those with iron, copper or cobalt as the central metal cation were not. Virus kill was not studied.
Singer et al (C. R. J. Singer, T. Azim and Q. Sattentau, "Preliminary Evaluation of Phthalocyanine Photosensitization For Inactivation Of Viral Pathogens in Blood Products", [abstract] British J. HematologyMar. 23-25, 1988: Abs. 31), in what is believed to be the only study on virus kill performed with phthalocyanines, demonstrated that an unstated quantity of both Epstein Barr virus and of HIV added to plasma was inactivated on treatment with 5 and 25 .mu.g/mL of sulfonated aluminum phthalocyanine and 2mW/cm.sup.2 for 30 minutes (3.6 J/cm.sup.2). Factor VIII recovery was only 50%. Singer et al reported no actual work on cells or cell-associated virus, though they state that application to red cells is being evaluated. Given the relatively poor recovery (50%) of factor VIII reported by Singer, the greater fragility of cells than proteins, and the previous experience on the photohemolysis of red cells to treatment with phthalocyanine, the results herein are all the more surprising.
Other Lipophilic Dyes in the Treatment of Whole Blood or Red Blood Cell Concentrates
Cole et al (Cole, M., Stromberg, R., Friedman, L., Benade, L., Shumaker, J., "Photochemical Inactivation of Virus in Red Cells", Transfusion, 29, Supp:42s Abs., 1989) explored the use of merocyanine 540 in the treatment of packed red blood cells diluted to a hematocrit of 15%. When plasma was removed such that its concentration was 2.6%, a 6 log reduction of vesicular stomatitis virus was achieved. However, only a 1 log reduction in VSV was achieved in samples containing 15% plasma. The authors concluded that "although plasma is required to protect the red blood cells from damage, viral kill is also significantly reduced". This conclusion is supported by the observation that the presence of 5% albumin inhibited virus kill in suspension of washed platelets (Prodouz, K. N., "Effect of Merocyanine 540 on Platelet Function and Reduction of its Antiviral Activity by Albumin", Transfusion, 29, Supp:42s Abs., 1989), and that though 6 logs of model viruses in buffer could be inactivated by merocyanine 540, only 1-3 logs of virus could be inactivated in the presence of 12-25% plasma (Moroff, G., Benade, L. E., Dabay, M., George, V. M., Shumaker, J. and Dodd, R. Y., "Use of Photochemical Procedures to Inactivate Viruses in Platelet Suspensions", Transfusion, 29, Supp:S15 Abs., 1989). Furthermore, the authors stated that the procedure "adversely affected platelet properties".
Matthews, J. T., Newman, J. T., Sogandares-Bernal, F., et al, "Photodynamic Therapy of Viral Contaminants with Potential For Blood Banking Applications", Transfusion, 1988;28:81-83 studied treatment of whole blood with hematoporphyrin derivatives and light. They reported the inactivation of 3.times.10.sup.5 PFU of herpes simplex virus type 1 (HSV-1) on treatment of culture medium with 2.5 .mu.g/mL dihematoporphyrin ether (DHE) and light, but only the inactivation of 10.sup.3 PFU on treatment of blood under the same condition. Increasing the concentration of DHE to 20 .mu.g/ml did not improve virus kill. While red blood cell structure and function was well maintained at 2.5 .mu.g/mL DHE and light at 5 J/cm.sup.2, cell-free HIV (2.times.10.sup.3 ID.sub.50) added to buffer alone was not completely killed under this condition.
Other Photoactive Compounds
Lin et al (Lin, L., Wiesehahn, G. P., Morel, P. A. and Corash, L., "Use of 8-Methoxypsoralen and Long-wavelength Ultraviolet Radiation for Decontamination of Platelet Concentrates", Blood, 74:517-525, 1989) demonstrated that psoralen and exposure to UV-A inactivated .gtoreq.10.sup.5.5 ID of feline leukemia virus added to a platelet concentrate; however, studies in whole blood or red cell concentrates were not performed. Platelet morphology, aggregation, and the release reaction were well maintained immediately following treatment, and showed comparable values when compared to untreated controls on storage for up to 96 hours. In contrast, Moroff et al (Moroff G., Benade, L.., Dabay, M., George, V. M., Shumaker, J. and Dodd, R. Y., "Use of Photochemical Procedures to Inactivate Viruses in Platelet Suspensions", Transfusion, 29, Supp, S15 Abs., 1989) explored the use of psoralen for the treatment of platelets and concluded that the presence of as little as 12% plasma inhibited virus kill and that platelet properties were adversely affected. It should be pointed out that, as typically prepared, red blood cells and platelet concentrates for transfusion are suspended in 100% plasma.
Other Agents
In U.S. patent application Ser. No. 07/279,179, filed Dec. 2, 1988, and in a recent published abstract (Williams et al, Blood, 1988, 72:Suppl.:287a), vesicular stomatitis virus added to whole blood was reported to be inactivated on incubation with a hydrolyzable, aryl diol epoxide without causing red cell lysis.
Ozone has been asserted to decontaminate whole blood containing 10.sup.9 pFU/mL of hepatitis virus (Zee, Y. C. and Bolton, D. C., "Ozone Decontamination of Blood and Blood Products", U.S. Pat. No. 4,632,980). However, no data were provided in support of this allegation. Prodouz, K. N., Fratantoni, J. C., Boone, J. E. and Bonner R. F., "Use of Laser-UV for Inactivation of Virus in Blood Products", Blood, 1987; 70:589-592 reported that laser-UV treatment of a platelet concentrate largely maintained platelet function under conditions where up to 10.sup.6 (ID.sub.50) of polio virus was inactivated. However, virus inactivation was studied in buffered medium alone and not in the presence of platelets, and only a cell-free form of the virus was employed.
Hartman et al (Hartman, F. W., Mangun, G. H., Feeley, N., Jackson, E., "On the Chemical Sterilization of Blood and Blood Products", Proc. Soc. Exp. Biol. Med., 70:248-254, 1949) showed that treatment of whole blood with the nitrogen mustard, methyl-bis(beta-chloroethyl) amine hydrochloride resulted in the inactivation of 10.sup.6.6 ID.sub.50 of vesicular stomatitis virus under conditions where red cell hemolysis was not greater than the control. It should be pointed out that nitrogen mustards are carcinogens.
LoGrippo (LoGrippo, G. A., "Investigations of the Use of Beta-Propiolactone in Virus Inactivation", Ann. NY Aca. Sci., 83, 578-594, (1959)) treated red cells separately from plasma with beta-propiolactone. Treatment resulted in the inactivation of more than 10.sup.8 ID.sup.50 of Eastern equine encephalitis virus without causing red cell hemolysis. Subsequent injection of the treated red cells in man resulted in a shortened circulatory half-life.